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Manish Mishra, Renu A. Kowluru; A novel regulator of oxidative stress in the development of diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3543.
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In the development of diabetic retinopathy, Rac1, a small molecular weight GTPase, is activated, which subsequently increases NADPH-oxidase 2 (Nox2)-mediated cytosolic reactive oxygen species (ROS) production. Activation of Rac1 is regulated by guanine exchange factors (GEFs), and Son of Sevenless 1 (Sos1) is one such GEFs implicated in its activation. Rac1-specific GEF activity of Sos1 is regulated by an adapter protein, p66Shc; destabilization of Sos1 binding to another adaptor protein, growth factor receptor bound protein (grb2), by p66Shc is shown to release Sos1 and activate Rac1. Our aim is to examine the role of p66Shc in the activation of Rac1 in the development of diabetic retinopathy.
Human retinal endothelial cells (HRECs), incubated in high glucose for 96 hours, were analyzed for p66Shc, Sos1, and Grb2. Role of p66Shc in Rac1 regulation was confirmed in p66Shc-siRNA transfected HRECs, and Rac1 activity was quantified by G-LISA colorimetric assay. Grb2 interaction with p66Shc, and with Sos1, was quantified by co-immunoprecipitation technique, and cellular co-localization of Sos1, p66Shc, and Rac1 was determined by immunofluorescence imaging.
High glucose increased p66Shc expression, and p66Shc-siRNA ameliorated glucose-induced Rac1 activation. However, high glucose had no effect on Sos1 and Grb2, and this was also confirmed by immunofluorescence imaging. Although glucose increased the binding of p66Shc with Grb2, the binding of Grb2 with Sos1 was decreased.
Thus, in hyperglycemic milieu, p66Shc inhibits Sos1-Grb2 binding, and increased GEF activity of Sos1 activates Rac1-Nox2-ROS signaling. This suggests that p66Shc has a major role in modulating ROS accumulation and the development of diabetic retinopathy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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